The major emphasis of this project is on synthesis of mechanism-based active site inhibitors of key enzymes, with special regard for development of potential cancer chemotherapy agents, as well as using these inhibitors to elucidate enzyme mechanisms. Attention is being focused on glutamine synthetase, aspartate transcarbamyolase, asparagine synthetase, and aspartokinase. Inhibitors synthesized and studied are generally either inert analogs of putative tightly bound transition states and unstable intermediates or suicide inactivators of these enzymes. Techniques employed include organic synthesis; steady state, rapid, and equilibrium isotope exchange kinetics; optical and magnetic resonance spectroscopy. Studies are also underway on the mechanism of activation of glutamine synthetases from plants and mammals by divalent cations, equilibrium exchange studies of modifier action, and thermophilic proteins.